The Runx1-CBFbeta transcription factor is required for the emergence of all definitive hematopoietic cells. It is the earliest specific marker of sites from which hematopoietic stem and progenitor cells are produced in the conceptus. Runx1 is expressed in endothelial cells, mesenchymal cells, and in intra-aortic hematopoietic clusters, and in all committed hematopoietic progenitors and transplantable stem cells. We hypothesize that Runx1-CBFbeta is required, at least in part, for the transition between hemogenic endothelium and hematopoietic cells during fetal development. However, if Runx1 is deleted in adult mice, after hematopoiesis is established, long-term repopulating hematopoietic stem cells persist, committed erythroid, myeloid, and megakaryocytic progenitors increase in number, and both erythropoiesis and terminal granulocyte differentiation are normal. Thus, there is an absolute requirement for Runx1-CBFbeta to establish hematopoiesis in the fetus, but not to maintain all aspects of it in the adult. One of our goals is to define the developmental window and cell types in which Runx1-CBFbeta is required to specify definitive hematopoiesis in the conceptus. A second aim follows up on recent findings that the placenta may be a source of hematopoietic stem cells. We will determine where in the placenta hematopoietic stem cells reside, and whether they differentiate from endothelial cells in the allantois or in the placental labyrinth. Finally, we will attempt to identify the signaling sources that induce the first wave of Runx1 expression and definitive hematopoiesis in the conceptus. We will specifically examine whether Hedgehog, the upstream component of a signaling cascade required for definitive hematopoiesis in zebrafish, activates Runx1 expression and definitive hematopoiesis in the mouse conceptus. Together these aims will contribute to our understanding of the earliest events that establish definitive hematopoiesis in the mouse conceptus, and should help guide efforts to produce blood cells from embryonic sources in vitro.